Triacylglycerol (TAG) is a universal storage lipid in plants, algae, fungi, and animals. TAG is composed of a glycerol backbone to which three fatty acyl chains are esterified. By transesterification with methanol, TAG can be converted into fatty acid methylesters (FAMEs) commonly referred to as biodiesel (Durrett et al. 2008). Microalgae have been considered as sustainable feedstock for the production of biofuels because they accumulate substantial amounts of TAG following nutrient deprivation. Theoretical calculations suggest that microalgae can surpass crop plants in their TAG yield per land area used (Weyer et al. 2010). Despite the recent interest in microalgae, this phylogenetically diverse group of photosynthetic organisms is not well understood at the molecular and biochemical levels, and the mechanistic basis of algal lipid metabolism and of TAG accumulation still needs to be explored in detail. Much of the current molecular understanding of photosynthetic lipid biosynthesis is based on work with Arabidopsis thaliana and other land plant models, providing paradigms that may not be directly transferable given their evolutionary divergence from microalgae. Indeed, current information on lipid metabolism in the green algal model Chlamydomonas reinhardtii, which is mostly based on genome annotation (Riekhof et al. 2005) or early labeling and lipid profiling experiments (Giroud et al. 1988, Giroud and Eichenberger 1989), suggests that lipid metabolism in this organism is distinct in crucial aspects from that of land plants. Most strikingly, Chlamydomonas lacks phosphatidylcholine (PtdCho), but instead contains the betaine lipid diacylglycerol-N,N,N,-trimethylhomoserine (DGTS).
Seed plants typically have two assembly pathways for glycerolipids (Roughan and Slack 1982). Fatty acids are synthesized de novo in the plastid while attached to acyl carrier proteins (ACPs) (Ohlrogge et al. 1979). Acyltransferases at the inner chloroplast envelope membrane transfer acyl groups from acyl-ACPs to glycerol 3-phosphate leading to the formation of phosphatidic acid (PtdOH), the precursor of glycerolipids of the thylakoid membrane. Alternatively, fatty acids are exported from the plastid for assembly of extraplastidic glycerolipids including TAGs at the endoplasmic reticulum (ER). Because the acyltransferases associated with the inner plastid envelope membrane and the ER have different acyl group preferences, glycerolipids assembled by the two pathways can be distinguished based on their acyl group composition (Heinz and Roughan 1983). In Chlamydomonas, the analysis of the acyl groups in the glyceryl backbone of the galactoglycerolipids, which are the predominant lipids in the thylakoid membranes, suggests that their assembly is completely dependent on the plastid pathway (Giroud et al. 1988). In contrast, in seed plants such as Arabidopsis the galactoglycerolipid molecular species are nearly equally derived from the ER and the plastid assembly pathway (Browse et al. 1986), thus requiring an elaborate system of lipid transfer between the ER and the plastid envelopes (Benning 2009).
In particular, the lack of phosphatidylcholine (PtdCho) in Chlamydomonas is expected to affect other aspects of glycerolipid metabolism. For example, isotope labeling of cytosolic lipids in pea leaves indicated that most of the acyl groups synthesized de novo in the plastid are first incorporated into PtdCho instead of PtdOH (Bates et al. 2007). Thus, it was proposed that acyl editing of PtdCho is an important aspect of fatty acid export from the plastid, cycling acyl groups through PtdCho before they enter the cytosolic acyl-CoA pool, which ultimately provides acyl groups for glycerolipid assembly at the ER. The lack of PtdCho in Chlamydomonas raises several questions, particularly whether an alternative mechanism of acyl editing, possibly involving DGTS or another lipid, or a mechanism completely independent of acyl editing exists, which is involved in the export of fatty acids from the plastid. Typically, lipid droplets are formed at the ER in all eukaryotic cells. However, recent reports on TAG accumulation in Chlamydomonas suggest that TAG-containing lipid droplets are present in plastids (Fan et al. 2011, Goodson et al. 2011), raising the possibility that TAG is either directly assembled in plastids, or imported into them.
Aside from the basic mechanisms of glycerolipid assembly in Chlamydomonas, the details of the regulation of TAG synthesis are unclear as well. Like other microalgae, Chlamydomonas produces lipid droplets filled with TAGs following nutrient deprivation (Moellering and Benning 2010, Wang et al. 2009), conditions that involve genome-wide transcriptional changes (Castruita et al. 2011, Miller et al. 2010). Intriguingly, among the genes up-regulated or down-regulated by N deprivation were a large number of genes annotated to encode lipases (Miller et al. 2010).